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US 2002/0172705 A1Published application
Bioengineered tissue constructs and methods for producing and using thereof
AssigneeOrganogenesis, Inc.
InventorsMichael P. Murphy, Vincent Ronfard
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Abstract
Cultured tissue constructs comprising cultured cells and endogenously produced extracellular matrix components without the requirement of exogenous matrix components or network support or scaffold members. Some tissue constructs of the invention are comprised of multiple cell layers or more than one cell type. The tissue constructs of the invention have morphological features and functions similar to tissues their cells are derived and their strength makes them easily handleable. Preferred cultured tissue constructs of the invention are prepared in defined media, that is, without the addition of chemically undefined components.
Claims30
A cultured tissue construct comprising fibroblasts cells grown under conditions to produce a layer of extracellular matrix which is synthesized and assembled by the cultured fibroblast cells, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) fibrillar collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) decorin;
- and,
- (iii) glycosaminoglycans;
- and wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions.
2 more elements
A cultured tissue construct comprising cultured dermal fibroblasts cells cultured under conditions to produce a layer of extracellular matrix which is synthesized and assembled by the cultured fibroblast cells, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) fibrillar type I and type III collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) decorin,
- (iii) fibronectin
- (iv) tenascin;
- and,
- (v) glycosaminoglycans;
- and wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions.
4 more elements
A cultured tissue construct having at least two layers, comprising:
- (a) a first layer of cultured fibroblasts cells cultured under conditions to produce a layer of extracellular matrix which is synthesized and assembled by the cultured fibroblast cells, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) fibrillar collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) decorin;
- and,
- (iii) glycosaminoglycans;
- wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions;
- and,
- (b) a second layer of cells comprising epithelial cells disposed on the first layer.
5 more elements
A cultured skin construct having at least two layers, comprising:
- (a) a first layer of cultured dermal fibroblasts cells cultured under conditions to produce a layer of extracellular matrix which is synthesized and assembled by the cultured fibroblast cells, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) type I and type III collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) decorin;
- (iii) fibronectin,
- (iv) tenascin;
- and,
- (v) glycosaminoglycans;
- wherein said extracellular matrix is produced by the cultured dermal fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions;
- and,
- (b) a second layer of keratinocyte cells disposed on the first layer to form an epidermal cell layer, wherein the epidermal cell layer is a multilayered, stratified, differentiated and exhibits a basal layer, suprabasal layer, a granular layer and a stratum corneum;
- and wherein the bilayered cultured skin construct has a basement membrane present at the junction of the first and second layers.
8 more elements
A method for producing a cultured tissue construct, comprising,
- (a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium;
- (b) culturing the fibroblast cells of step (a) in the first cell culture medium to between about 80% to about 100% confluence on the porous membrane;
- (c) stimulating the fibroblast cells to synthesize, secrete and organize extracellular matrix components under culturing conditions in a second culture medium;
- and,
- (d) continued culturing of the fibroblast cells until the cells form a layer of synthesized extracellular matrix of at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) fibrillar collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (it) decorin;
- and,
- (iii) glycosaminoglycans;
- and wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions.
7 more elements
A method for producing a bilayered cultured tissue construct, comprising:
- (a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium;
- (b) culturing the fibroblast cells of step (a) in the first cell culture medium to between about 80% to about 100% confluence on the porous membrane;
- (c) stimulating the fibroblast cells of step (a) to synthesize, secrete and organize extracellular matrix components under culturing conditions in a second culture medium;
- (d) continued culturing of the fibroblast cell until the cells form a layer of synthesized extracellular matrix of between about 30 to about 110 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) fibrillar collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) decorin;
- and,
- (iii) glycosaminoglycans;
- and wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions;
- (e) seeding epithelial cells to the top surface the synthesized extracellular matrix of step (d), and,
- (f) stimulating the epithelial cells of step (e) under culturing conditions to form a bilayered tissue construct of an extracellular matrix, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, and a second layer of epithelial cells.
8 more elements
A method for producing a bilayered cultured skin construct comprising a dermal layer and an epidermal layer disposed thereon in the absence of a structural support scaffold or exogenous matrix components, wherein the method comprises the steps of:
- (a) seeding fibroblast cells on porous membrane in a culture vessel in a first chemically defined cell culture medium at a density between about 1×105 cells/cm2 to about 6.6×105 cells/cm2;
- (b) culturing the fibroblast cells of step (a) in the first chemically defined cell culture medium to between about 80% to about 100% confluence;
- (c) stimulating the fibroblast cells of step (a) to synthesize, secrete and organize extracellular matrix components by culturing the cells in a second chemically defined culture medium;
- (d) continued culturing of the fibroblast cells until the cells form a layer of synthesized extracellular matrix of at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer,
- wherein the extracellular matrix comprises:
- (i) type I and type III collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) decorin;
- (iii) tenascin;
- and,
- (iv) glycosaminoglycans;
- wherein said extracellular matrix is produced by the cultured dermal fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions;
- (e) seeding keratinocyte cells to the top surface of the synthesized extracellular matrix of step (d), and,
- (f) culturing the keratinocyte cells under culturing conditions to form an epidermal layer,
- wherein the epidermal cell layer is a multilayered, stratified, differentiated layer of keratinocytes that exhibit a basal layer, a suprabasal layer, a granular layer and a stratum corneum;
- and wherein the bilayered cultured skin construct has a basement membrane present at the junction of the first and second layers.
12 more elements
A method for producing a cultured tissue construct, comprising,
- (a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a cell culture medium at about 80% to about 100% confluence;
- (b) stimulating the fibroblast cells to synthesize, secrete and organize extracellular matrix components under culturing conditions in a second culture medium;
- and,
- (c) continued culturing of the fibroblast cells until the cells form a layer of synthesized extracellular matrix of at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises:
- (i) fibrillar collagen showing a packing organization of fibrils and fibril bundles exhibiting a quarter-staggered 67 nm banding pattern;
- (ii) tenascin;
- and,
- (iii) glycosaminoglycans;
- and wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions.
6 more elements
Dependencies are inferred from the claim text.